Conservation of Threatened Plants of India
In vitro propagation of some threatened plant species of India C. R. Deb1,*, G. R. Rout2, A. A. Mao3, S. K. Nandi4, R. K. Nilasana Singha3, D. Vijayan3, T. Langhu1, Z. P. Kikon1, S. Pradhan3, Mohd Tariq4 and D. Swain2 1
Department of Botany, Nagaland University, Lumami 798 627, India Department of Agricultural Biotechnology, College of Agriculture, Orissa University of Agriculture and Technology, Bhubaneswar 751 003, India 3 Botanical Survey of India, Eastern Regional Centre, Shillong 793 003, India 4 G.B. Pant Institute of Himalayan Environment and Development, Kosi-Katarmal, Almora 263 643, India 2
Keywords: Conservation, in vitro regeneration, microshoots, threatened species.
the establishment of extensive collections using minimum space, and allow supply of valuable material for recovery of wild threatened species populations10. The present study was designed to demonstrate that the threatened species facing regeneration failure in nature and which are difficult to propagate through other costeffective conventional vegetative and sexual propagation methods are best suited for in vitro propagation. We developed efficient protocols for in vitro plant regeneration of Aconitum nagarum Stapf, Hypericum gaitii Haines, Podophyllum hexandrum Royale, Rhododendron macabeanum Watt ex Balf. f., Rhododendron wattii Cowan and Vanda bicolor Griff. using different explants and manipulation of plant growth regulators and culture conditions.
Introduction
Materials and methods
CONSERVATION of plant genetic resources can be achieved in situ as well as ex situ1. Both cultivated and domesticated plant species are also maintained in their natural habitats as well as in field conditions2,3. Due to habitat destruction and transformation of the natural environment, several species have been lost from the ecosystems. Therefore, in situ methods alone are insufficient for conserving the threatened species. Under these circumstances, ex situ conservation is a viable alternative for preventing extinction of threatened species. In some cases, it is the only viable strategy to conserve certain species. In situ and ex situ methods are complementary and not mutually exclusive. Selection of appropriate strategy should be based on a number of criteria including the status of the species and feasibility of applying the chosen methods4. In vitro culture method is a powerful tool for propagation, conservation and management of commercially important and threatened plant species5–9. In vitro culture technique has been used for large-scale propagation of threatened species, thus improving the conservation status of the species. Further, in vitro techniques offer a safe way for international exchange of plant materials, enable
Study species
To prevent extinction of threatened species, in vitro regeneration protocols for the propagation of six threatened species were standardized. The regenerated micro-shoots were rooted in nutrient medium supplemented with low concentrations of auxin. The well-developed plantlets were successfully established in field conditions, thus improving the probability of self-sustenance of the introduced populations. The success story of these six threatened species reaffirms the role of in vitro propagation in conserving plants facing the threat of extinction.
*For correspondence. (e-mail:
[email protected]) CURRENT SCIENCE, VOL. 114, NO. 3, 10 FEBRUARY 2018
Aconitum nagarum Stapf (Ranunculaceae): The genus Aconitum is represented by nearly 300 species in the world, of which India has 33 species. A. nagarum is a herb that grows at altitudinal range of 1600–3800 m amsl. The alkaloids produced from its rhizomes are used for cure of a wide range of ailments, and are used as arrow poison. The plant has antibacterial properties against Staphylococcus aureus, Salmonella typhimutium, Escherichia coli and Bacillus subtilis3. Owing to its high medicinal value, A. nagarum is being exploited from its natural habitat and thus has become threatened. Hypericum gaitii Haines (Hypericaceae): The genus Hypericum is represented by 494 species, of which 29 species occur in India11. Among these 29 species, three, viz. H. assamicum, H. gaitii and H. gracilipes are endemic to India12. Hypericum species can be used in the treatment of cancer and AIDS. It is popular as an antidepressant13. H. gaitii is a tall shrub, bushy with erect branches, and is a threatened species in the Eastern Ghats region of India owing to habitat loss and different anthropogenic activities. There is a preliminary report on in vitro axillary shoot multiplication of H. gaitii14. 567
Special Section: However, no report is available on direct plant regeneration from leaf and stem explants of H. gaitii. Podophyllum hexandrum Royle (Podophyllaceae): P. hexandrum is a perennial threatened herbaceous plant species with a wide distribution range, i.e. 2000– 4000 m amsl in the Indian Himalayan Region 15. It is a source of anti-tumour drugs, viz. etoposide, etopophos and teniposide16,17. The natural populations of P. hexandrum are low and steadily declining due to high demand of its rhizomes. P. hexandrum has a relatively long juvenile phase18. Propagation of the species is undertaken through vegetative means and seeds. Natural regeneration is poor due to erratic seed-setting, long seed dormancy (1–2 years) as an adaptation strategy to overcome harsh climatic conditions, and trampling by grazing animals. Hence, in vitro techniques can be an alternate and effective means of propagation 19. Moreover, considering its medicinal importance, in vitro method offers stablility in bioactive molecule production. Rhododendron macabeanum Watt ex Balf.f. and R. wattii Cowan (Ericaceae): Rhododendron is the largest woody plant genus in Ericaceae, represented by 1025 species in the world20. In India, 135 species of rhododendrons have been recorded, among which 132 species are from North East (NE) India region (unpublished data). R. wattii and R. macabeanum are endemic to Manipur and Nagaland, and the natural populations are scarce21. Anthropogenic activities like extraction for firewood and natural calamities such as forest fire during the dry season might have contributed to the rapid disappearance of these species. Considering the failure of both the species to regenerate through seeds, there is an urgent need to establish a suitable protocol for in vitro propagation22–24 . Vanda bicolor Griff. (Orchidaceae): V. bicolor is a horticulturally important monopodial orchid reported only in NE India, Bhutan, Myanmar and Nepal25. Due to habitat destruction, the species is now threatened. Considering its economic importance and poor natural regeneration, the present study was undertaken to develop an efficient protocol for mass propagation of the species for its commercial exploitation as well as conservation in the wild.
Plant material collection and culture condition Aconitum nagarum: Matured fruits were collected during October and November in 2014 and 2015 from Khonoma village (Dzuko valley, altitude 2684 m amsl), Nagaland. Seeds were surface-sterilized with aqueous solution of HgCl 2 (0.2%, w/v) for 5 min, and subsequently rinsed 4–5 times with sterile pure water. Sterilized seeds were soaked in sterilized water. Shoot buds were collected from germinated seedlings grown during different seasons used as a source of explants. 568
Hypericum gaitii: In vitro shoot multiplication via shoot tip culture was established as reported by Swain et al.25. Both young leaves (0.5 cm2 ) and internodal segments (0.25–0.5 cm) were excised from 8-week-old in vitro grown shoots, and were used as a source of explants for direct shoot bud regeneration without the intervening callus phase. The cultures were maintained in an incubation room with 16 h photoperiod and 3000 lux light intensity at 25 2C. Podophyllum hexandrum: Matured fruits were collected during April and September in 2013 and 2014 from Martoli village region (3438 m amsl) of Pithoragarh district, Uttarakhand. Seeds were extracted, washed and treated with Bavistin (0.2%, w/v) for 10 min. They were dried under shade and stored at 4C till use. The seeds were sterilized with HgCl2 (0.04%) for 8 min and washed four times with sterilized distilled water. Subsequently, they were scarified for 3 min with H2 SO4 (50%, v/v) before culture on nutrient medium. Rhododendron macabeanum and Rhododendron wattii: Matured seeds of both the species were collect during December 2015 from Dzukou valley (2575 m amsl), Nagaland. Seeds were washed with Tween-80 (1.0%, v/v) for ~5 min, and subsequently washed five times in sterile distilled water before culturing on agar gel Anderson medium (AM) enriched with sucrose (3%) for germination. The cotyledonary node and shoot tip explants obtained from in vitro derived seedlings were used for multiple shoot induction. Vanda bicolor: Immatured seed pods of V. bicolor were collected during July 2014. Seed pods were surface cleansed with detergent (Labolene, India) and rinsed under running tap water. Surface-washed green pods were treated with HgCl2 (0.2%) for 5 min followed by washing 4–5 times with sterile pure water. The pods were then dipped in ethanol (70%, v/v) and flamed before scooping out the seeds.
Initiation of culture Cultures were initiated for all the species from different explant sources. For this, different nutrient media fortified differently were used. Tables 1 and 2 provide details of explant type(s), nutrient medium, supplements, plant growth regulators (PGRs) and culture condition(s). The multiple shoots/regenerated propagules developed on initiation medium were transferred to fresh medium either of same composition or of different combinations for multiplication.
Plant regeneration/multiple shoots induction, rooting, hardening and transplantation of regenerates For shoot multiplication and regeneration, rooting and hardening of regenerates derived from different protocols were followed (Table 3). CURRENT SCIENCE, VOL. 114, NO. 3, 10 FEBRUARY 2018
Conservation of Threatened Plants of India Table 1. Species Aconitum nagarum Hypericum gaitii
Podophyllum hexandrum Rhododendron macabeanum Rhododendron wattii Vanda bicolor
Explants, nutrient media and supplements used for initiation of in vitro culture of different species
Explants used for culture initiation Shoot buds from in vitro grown seedlings Leaf, internodal segments from in vitro grown plantlets Mature seeds Shoot apices and nodal segments from in vitro grown seedlings Nodal segments from in vitro grown seedlings Immature embryos
Basal medium and supplements
Culture conditions
MS medium + sucrose (3%) + BA (3–15 M) and NAA (1–2 M) singly or in combination MS medium + sucrose (3%) + kinetin and BA (0.5–1.5 mg l–1 ) and NAA (0.25–0.50 mg l–1 ), either singly or in combination MS medium + sucrose (3%) + GA3 (0.1 M) and BA (1.0 M) in combination AM, modified AM and WPM + sucrose (3%, w/v) + 2iP, BA and kinetin (1–8 mg l–1 ) separately
40 mol m–2 s–1 illumination with 12 h photoperiod at 25 2C 55 mol m–2 s–1 light intensity with 16 h photoperiod 25 2C 42 mol m–2 s–1 illumination with 16 h photoperiod at 25 2C White fluorescent light with a 16 h photoperiod at 25 2C
AM, modified AM and WPM + sucrose (3%, w/v) + 2iP and BA (1–8 mg l–1 ) separately 40 mol m–2 s–1 illumination with 12 h photoperiod at 25 2C
MS medium + sucrose (3%) + NAA and BA (3–9 M) either singly or in combination
MS: Murashige & Skoog; BA, Benzyl adenine; NAA, -naphthaleneacetic acid; WPM, Woody plant medium; 2iP, N6(2-iso pentenyl) adenine; GA, Gibberellic acid; AM, Agar medium.
Table 2. Effects of nutrient media, plant growth regulators (PGRs) and adjuncts on in vitro morphogenetic response of explants of different species Plant species
Optimum medium, PGRs and adjuncts
Morphogenetic pathway
A. nagarum H. gaitii
MS medium + sucrose (3%) + BA (6 M) MS medium + sucrose (3%) + BA (1.0 mg l–1 )
Shoot buds Direct shoot bud regeneration
P. hexandrum
MS medium + sucrose (3%) + GA3 (0.1 M) and BA (1.0 M) in combination WPM + sucrose (3%) + 2iP (4 mg l–1) WPM + sucrose (3%) + 2iP (8 mg l–1) MS medium + sucrose (3%) + NAA and BA (3 M in combination)
R. macabeanum R. wattii V. bicolor
No. of propagules developed per explant
% Response
16 86 (internodal segment), 56 (leaf explants)
Seedlings
95 85.6 (internodal segment), 73.8 (leaf) –
Shoot buds Shoot buds PLBs, plantlets
76 – 88
8 7 –
–
PLBs, Protocorm like bodies; other abbreviations are same as Table 1.
Aconitum nagarum: The micro-shoots developed in the initiation medium were transferred to the same medium for another 2–3 passages. In every subculture, the microshoots formed were separated and transferred to fresh regeneration medium for further proliferation. About 5– 6 cm long micro-shoots with well-expanded leaves were selected for inducing roots. They were subjected to acclimatization and transplantation of the regenerates was carried out in the potting mix. The micro-shoots were maintained on MS medium containing NAA (0–5 M) and maintained at 16 h photoperiod at 25 2C. The well-rooted plantlets were taken out from the rooting medium and transferred to MS medium fortified with sucrose (3% w/v) devoid of PGRs and maintained in the laboratory for 3–4 weeks. The hardened plantlets were separated from the culture vials and washed with tap water. They were then transplanted on plastic pots containing a mixture of soil, decayed wood powder and coconut coir in the ratio 1 : 1 : 1. The pots were covered with holed transparent polybags and kept moist through CURRENT SCIENCE, VOL. 114, NO. 3, 10 FEBRUARY 2018
capillary water for 2 weeks. The acclimatized transplants were finally transferred to the greenhouse. Hypericum gaitii: Leaf and internodal segments derived from in vitro raised shoots of H. gaitii were cultured on semi-solid basal MS medium supplemented with 3% (w/v) sucrose and different concentrations and combinations of BA (0.0–1.5 mg l–1) or kinetin (0.0–1.5 mg l–1 ), NAA (0.0–0.5 mg l –1) for direct shoot-bud regeneration. The regenerated micro-shoots were maintained in a medium with similar composition at every 4 weeks interval. Fifteen replicates were employed for each treatment and the experiment was repeated four times. After 8 weeks, the regenerated micro-shoots (~2–3 cm length) were separated from the culture medium and transferred to half-strength MS semi-solid medium with 2% (w/v) sucrose and different concentrations of indole-3-acetic acid (IAA) or indole-3-butyric acid (IBA) (0.0–1.0 mg l–1) for induction of roots. Rooted plantlets were transferred to plastic cups containing sterile distilled water for 569
Special Section: Table 3.
Nutrient media, growth supplements and culture conditions for culture proliferation, rooting, hardening and transplantation of regenerates
Species
Optimum shoot proliferation medium and adjuncts
Rooting medium
Hardening
A. nagarum
MS medium + sucrose (3%) + BA (6 M)
MS medium + sucrose (3%) + NAA (5 M) for 8 weeks
MS medium + sucrose (3%) for 4–6 weeks
H. gaitii
MS medium + sucrose (3%) + BA (1.0 mg l–1 )
MS medium + sucrose (2%) + IBA (1 mg l–1 ) for 2 weeks
P. hexandrum
MS medium + sucrose (3%) + GA3 (0.1 M) and BA (1.0 M) in combination. WPM + sucrose (3%) + 2iP (4 mg l–1 ) WPM + sucrose (3%) + 2iP (8 mg l–1 )
MS medium + sucrose (3%) + IAA (1.0 M)
Rooted plantlets maintained in plastic glass containing sterile distilled water for 2 weeks In polybags containing soil and farmyard manure (3 : 1, w/w)
MS medium + sucrose (3%) + NAA and BA (3 M each) + 0.6% AC
As in proliferation medium. Simultaneous rooting on multiplication medium
R. macabeanum R. wattii
V. bicolor
WPM + AC (0.2%, w/v)
WPM + AC (0.2%, w/v)
WPM + AC (0.2%, w/v)
Planted in root trainer filled with coarse sand and leaf moulds at 1 : 1 ratio and maintained in mist chamber Matrix of moss, charcoal pieces, brick pieces and decayed wood in the ratio 1 : 1 : 1 : 1 mixed with 1/10th of MS medium without sucrose and growth regulators for 5–6 weeks
Potting mix for transplantation Plastic pots containing soil : decayed wood powder : coconut coir (1 : 1 : 1 ratio, v/v). Transplants maintained under 75% shade for 4–6 weeks Polybags filled with garden soil, sand and cow dung at 2 : 1 : 1 ratio (v/v)
Polybags filled with soil and farmyard manure (3 : 1, w/w) for about 7–8 weeks Root trainer filled with coarse sand and soil at 1 : 1 ratio Polybags filled with coarse sand and leaf moulds in 1 : 1 ratio
Charcoal pieces, brick pieces and moss in 1 : 1 : 1 ratio. The transplants were maintained in polyhouse under 75% shade for 3–4 weeks
Incubation conditions for culture proliferation, rooting and hardening are the same as in the culture initiation shown in Table 1. AC, Activated charcoal; other abbreviations are same as Table 1.
primary acclimatization for 2 weeks in the growth chamber at 28C with 70% relative humidity and 16-h photoperiod. Further, the plantlets were transferred to polybags containing a mixture of sterile sand : soil : well rotted cow-dung manure (1 : 1 : 1). They were kept in the greenhouse for secondary acclimatization before transferred to the field. Watering was done daily to maintain humidity and moisture.
shoot induction. Shoots were subcultured at 20 days intervals. Ten replicates were employed for each treatment and the experiment was repeated four times. About 1.5– 2.0 cm long in vitro-raised shoots were transferred to liquid WPM medium using filter-paper bridge technique supplemented with different concentrations of IBA and naphthaleneacetic acid (NAA) (0.5–4.0 mg l–1) for induction of roots.
Podophyllum hexandrum: Prominent cotyledonary tube with multiple leaves produced from germinating seeds was maintained on the germination medium for 2–3 weeks. Shoots were rooted by culturing on MS medium containing 1.0 M IAA. Rooted plants were kept for hardening in polybags containing soil and farmyard manure (3 : 1, w/w) for about 7–8 weeks.
Rhododendron wattii: Nodal segment explants (1.5– 2.0 cm in length) from 2-month-old aseptic seedlings of R. wattii were cultured on different nutrient media (AM, modified AM and WPM) supplemented with different concentrations of BA and 2iP (1–12 mg l–1 ) and different additives (100 mg l –1 polyvinyl pyrrolidone (PVP), 100 mg l–1 ascorbic acid and citric acid 10 mg l–1 ) for multiple shoot induction. Cultures were sub-cultured at 3 week intervals. Individual shoots of ~1.5–2.0 cm length isolated from the shoot clump of in vitro culture were placed on AM, WPM containing activated charcoal (0.2%) and liquid medium using filter paper bridge technique supplemented with different concentrations of IBA and NAA (0.5–2.0 mg l–1) for induction of roots. The in
Rhododendron macabeanum: About 2–3-month-old in vitro raised seedlings were used as explants, viz. apical shoots, nodal segments, and roots for multiple shoot induction. Different concentrations (1–8 mg l –1) of N6 (iso pentyryl adenine), BA and kinetin (Kin) in woody plant medium (WPM) were investigated for multiple 570
CURRENT SCIENCE, VOL. 114, NO. 3, 10 FEBRUARY 2018
Conservation of Threatened Plants of India vitro grown plantlets were transferred to the greenhouse and planted in root trainer containing a mixture of coarse sand and leaf moulds (1 : 1) and the temperature of the greenhouse was maintained at 25C. Vanda bicolor: The PLBs and shoots developed from germinated seeds were transferred to MS medium supplemented with BA and NAA (0–9 M, either singly or in combination) and activated charcoal (AC) (0.3–0.9%, w/v), and maintained for 2–3 passages at 4 weeks interval for plant regeneration and multiplication. Plantlets with well-developed roots were separated from the clumps and transferred for hardening24. Plantlets were maintained under laboratory condition on a matrix of autoclaved moss, charcoal pieces, brick pieces and decayed wood in the ratio 1 : 1 : 1 : 1. MS nutrient medium (1/10th strength) without sucrose and growth regulators was applied for 5– 6 weeks. The primary hardened plantlets were transferred to pots with charcoal pieces, brick pieces and moss in the ratio of 1 : 1 : 1. The transplants were maintained in the polyhouse at 75% shade for 3–4 weeks before transferring to the field.
Results and discussion Cultures were initiated from different explants of various species as described above on different nutrient media
Figure 1. Different in vitro propagation stages of Aconitum nagarum. a, Excised shoot bud cultured on initiation medium showing signs of morphogenetic response by releasing the meristematic loci. b, Multiple shoots/micro-shoots developed in culture. c, Rooted plant. d, Regenerates transplanted in potting mix maintained in the polyhouse. CURRENT SCIENCE, VOL. 114, NO. 3, 10 FEBRUARY 2018
with manipulation of PGRs and additives. The explants exhibited differential responses (Table 2).
Aconitum nagarum Culture was initiated from the shoot buds harvested during different seasons cultured on MS medium fortified with different PGRs. Among the different levels of PGRs used, BA alone was found to be beneficial for culture initiation against the combined treatment of BA and NAA (Table 2). Under the conditions provided in the present study, optimum morphogenetic response was registered on medium supplemented with (3%) sucrose and BA (6 M). Within one week of culture, explants started responding by way of swelling and formation of loci (Figure 1 a). At lower concentration of BA, fewer shoot buds formed per explant cultured, while at higher concentration of BA delayed morphogenetic response was observed. Under optimum conditions, as many as 16 shoot buds were formed per explant in ~95% of culture (Figure 1 b). Different plants exhibited differential seasonal rhythm for growth and in vitro morphogenetic response. When BA-enriched medium was fortified with NAA, it supported lesser micro-shoot formation and there was callusing of the shoots at the basal part, exhibiting stunted growth. Earlier, Karuppusamy et al. 26 reported the synergistic effect of NAA and BA on nodal explants culture of Hydrocotyle conferta. Dhavala and Rathore27 reported that cytokinin alone could not promote axillary budbreaking in Embelia ribes, unless one of the auxins,
Figure 2. Direct plant regeneration of Hypericum gaitti. a, b, Proliferation of leaf (a) and intermodal explants (b) grown on MS medium supplemented with 1.0 mg l–1 BA after 4 weeks of culture. c, d, Development of shoots from intermodal and leaf explants on MS medium supplemented with 1.0 mg l–1 BA and 3% sucrose after 4 weeks of subculture. e, Elongation of shoots on MS liquid medium supplemented with 1.0 mg l–1 BA and 3% sucrose after 4 weeks of third subculture. f, Induction of roots from micro-shoots on half-strength MS medium supplemented with 1.0 mg l–1 IBA and 2% sucrose after three weeks of culture. g, In vitro raised plantlets acclimatized in distilled water and ready for transfer to greenhouse. h, In vitro raised plants grown in the green house after 2 months of transfer. 571
Special Section: especially IAA was incorporated in the medium in conjunction with cytokinin. While in guava nodal segment culture, incorporation of GA3 along with BA was the prerequisite for axillary bud-breaking28. However, in Adhatoda vasica, axillary bud proliferation and multiple shoot initiation were optimum on MS medium containing BA alone29.
maintained for longer period without any loss in the morphogenetic potential (Figure 2 e). The regenerative potential in both the leaf and internodal explants was higher under 16 h photoperiod than continuous light. The synergistic effect of photoperiod and growth regulators on in vitro shoot bud differentiation as observed in the present study was also noted in Lavandula latifolia33 and Prunus species30,34.
Hypericum gaitii Podophyllum hexandrum Direct initiation of morphogenetic response was observed from leaf and internodal segments, which subsequently developed into green, globular structures on the cut surfaces as well as above the explants on MS basal medium supplemented with 0.5–1.5 mg l–1 BA (Figure 2 a and b) followed by dark green shoot formation. The medium supplemented with kinetin alone or Kn + NAA or BA + NAA did not promote healthy morphogenesis. Healthy shoot-bud regeneration was observed on MS medium fortified with BA (1.0 mg l–1 ) (Figure 2 c and d). High regeneration frequency (~85.6%) was achieved in the internodal explants compared to leaves (~73.8%) on MS medium containing 1.0 mg l–1 BA (Table 2). Liu and Sanford30 made similar observations in strawberry in a medium containing BA in combination with IBA. Induction of direct shoot-bud regeneration in the medium having either higher (>1.5 mg l–1 ) or lower concentration (
